The mammalian immune system has evolved in order to survive in the environment containing a large variety of microorganisms, which colonize them in a number of niches like skin, intestine, upper and lower respiratory tract, urogenital tract etc. Some of the niches like colon and skin are colonized constitutively by an endogenous microbiota, whereas other niches (internal organs and lower respiratory tract) are normally kept sterile in an immunocompetent host. The effects of microorganism can be positive for the host, as is the case for the many intestinal symbiotic bacteria. In other cases, microbial colonization can be detrimental to the host. Such negative effects depend on the status of the host's immune system—certain pathogens (known as opportunistic pathogens) affect only immunocompromised individuals. The potential detrimental effect of microbial infections has led to the evolution of variety of host-defence mechanisms. In jawed vertebrates, there are two types of defence: innate and adaptive immune responses. The main distinction between these is the receptor types used to recognize pathogens, the time-delay needed to launch the response and the presence/absence of memory. The two types of defence do not operate completely independently from each other. As seen in the below, innate immune system sends specific signals to the adaptive immune system, helping to mount the response that is most efficient to the specific pathogen; and vice versa—adaptive immune response also activates some modules of the innate immune system.
Innate Immune Response
Innate immunity is always present in healthy individuals and its main function is to block the entry of microbes and viruses as well as to provide a rapid elimination of pathogens that do succeed in entering the host tissues. It provides immediate protection for the multicellular organism.
Innate immune system is not a single entity. It is a collection of distinct modules or subsystems that appeared at different stages of evolution:                Mucosal epithelia producing antimicrobial peptides, protecting the host from pathogen invasion;        phagocytes with their anti-microbial mechanisms against intra- and extracellular bacteria;        acute-phase proteins and complement system that are operating in the circulation and body fluids;        natural killer cells, which are involved in killing virus infected cells;        eosinophils, basophils and mast cells, which are involved against protection of multicellular parasites;        type I interferons and proteins induced by them, which have a crucial role in defence against viruses.        
The innate immune response is responsible for the early detection and destruction of invading microbes, and relies on a set of limited germ line-encoded pattern-recognition receptors (PRRs) for detection. To initiate immune responses, PRRs recognize pathogen-associated molecular patterns (PAMPs) and induce several extracellular activation cascades such as the complement pathway and various intracellular signalling pathways, which lead to the inflammatory responses.
The innate immune system utilizes PRRs present in three different compartments: body fluids, cell membranes, and cytoplasm. The PRRs in the body fluids play major roles in PAMP opsonization, the activation of complement pathways, and in some cases the transfer of PAMPs to other PRRs. PRRs located on the cell membrane have diverse functions, such as the presentation of PAMPs to other PRRs, the promotion of microbial uptake by phagocytosis, and the initiation of major signalling pathways.
There are several functionally distinct classes of PRRs. The best characterized class is Toll-like receptors (TLRs). These are transmembrane receptors that recognize viral nucleic acids and several bacterial products, including lipopolysaccharide and lipoteichoic acids and are the primary signal-generating PRRs (Akira, S 2006). In addition, cytoplasmic PRRs which can be grouped into three classes: interferon (IFN)-inducible proteins, caspase-recruiting domain (CARD) helicases, and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). Among the best studied IFN-induced antiviral proteins are the family of myxovirus resistance proteins (Mx), protein kinase R (PKR), oligoadenylate synthetase (2′-5′ OAS). These antiviral proteins and CARD helicases such as RIG-I and Mda5 are involved in antiviral defence. In contrast, NLRs are mainly involved in antibacterial immune responses.
Toll-Like Receptors (TLRs)
TLRs are the best-characterized signal-generating receptors among PRRs. They initiate key inflammatory responses and also shape adaptive immunity. All TLRs (TLR1-11) known in mammals are type I integral membrane glycoproteins containing an extracellular leucine-rich repeat (LRR) domain responsible for ligand recognition and a cytoplasmic Toll-interleukine-1 receptor homology (TIR) domain required for initiating signalling. TLRs recognize quite diverse microbial components in bacteria, fungi, parasites, and viruses including nucleic acids. Although normally present at the plasma membrane to detect extracellular PAMPs, a few TLRs, including TLR3, TLR7, TLR8, and TLR9, recognize their ligands in the intracellular compartments such as endosomes. The latter TLRs share the ability of nucleic acid recognition, detecting dsRNA (TLR3), ssRNA (TLR7 in mice, TLR8 in humans), and non-methylated CpG DNA motifs (TLR9).
TLRs initiate shared and distinct signalling pathways by recruiting different combinations of four TIR domain-containing adaptor molecules: MyD88, TIRAP, Trif, and TRAM. With the exception of TLR3, all the other TLRs recruit the myeloid differentiation factor 88 (MyD88), which is associated with members of the IL-receptor-associated kinase (IRAK) family (Mouldy Sioud 2006). These signalling pathways activate the transcription factors nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1), which is common to all TLRs, leading to the production of inflammatory cytokines and chemokines. They also activate interferon regulatory factor-3 (IRF3) and/or IRF7 in TLRs 3, 4, 7, 8, and 9 which is a prerequisite for the production of type I interferons such as IFN-α and IFN-β (For review Edwards et al 2007, Vercammen et al 2008, Medzitov R 2007).
In addition to direct activation of innate host-defence mechanisms, some PRRs are coupled to the induction of adaptive immune responses. T-and B-cells, the two main classes of cells in the adaptive immune system, express antigen binding receptors with random specificities and therefore recognize antigens that lack any intrinsic characteristics indicative of their origin. Therefore, T-and B-lymphocytes require instructions indicating the origin of the antigen they recognize. These instructions come from the innate immune system in the form of specialized signals inducible by PRRs. For T-cells this association is interpreted by dendritic cells. Type I interferons are involved in the activation and migration of dendritic cells (described in more details under Antiviral response). When activated dendritic cell migrates to the lymph node, they present the pathogen-derived antigens, together with PRR-induced signals, to T-cells. This results in T-cell activation and differentiation of T-helper (Th) cells into one of several types of effector Th-cells (Th1, Th2 and Th-17 cells). For instance TLR-engagement induces IL-12 production by dendritic cells, which directs Th cells to differentiate into Th1 cells. The type of effector response is thus dictated by the innate immune system. In addition, type I interferons also regulate the function of cytotoxic T-cells and NK cells, either directly or indirectly by inducing IL-15 production.
The innate immune system also receives positive feedback signals from the adaptive immune system. For instance, effector Th-cells produce appropriate cytokines that activate specific modules of the innate immune system: macrophages are activated by cytokines (interferon-γ) secreted from Th1 cells, neutrophils are activated by Th-17 cells (interleukin-17) cells, mast cells and basophils are activated by Th2 cells (interleukin-4 and -5). Likewise, bound antibodies (IgG) activate complement proteins and help phagocytosis by opsonizing pathogens.
Adaptive Immune Response
The adaptive immune system uses a broad range of molecules for its activities. Some of these molecules are also used by the innate immune system, e.g. complement proteins, others, including antigen-specific B-cell and T-cell receptors, are unique to the adaptive immune system. The most important properties of the adaptive immune system, distinguishing it from innate immunity, are a fine specificity of B- and T-cell receptors, and a more slow development of the response and memory of prior exposure to antigen. The latter property forms the basis of vaccination—priming of the immune system by attenuated pathogen, by selected components of the pathogen or by mimicking infection in other ways (e.g. by DNA-vaccine encoding selected antigens from a pathogen) results in the development of immunological memory, which triggers response more quickly and more efficiently upon pathogen encounter.
There are two types of adaptive immunity, humoral immunity and cell-mediated immunity. Humoral immunity is mediated by B-cells. Activated B-cells start to secrete the receptors into circulation and mucosal fluids, which in this case are referred to as antibodies (immunoglobulins). The genes encoding these receptors are assembled from variable and constant fragments in the process of somatic recombination, prior to pathogen encounter, which yields a diverse repertoire of receptors. Each B- or T-cell is able to synthesize immunoglobulins or T-cell receptors of a single specificity that bind to a specific molecular structure (epitope). Antibodies bind noncovalently to specific antigens to immobilize them, render them harmless or tag the antigen for destruction (e.g. by complement proteins or by macrophages) and removal by other components of the immune system. Cell mediated immunity is mediated by T-cells. T-cells are key players in most adaptive immune responses. They participate directly in eliminating infected cells (CD8+ cytotoxic T-cells) or orchestrate and regulate activity of other cells by producing various cytokines (CD4+ T-helper cells). Also the induction of antibodies by B-cells is in a majority of cases dependent on T-helper cells. The distinguished feature of T-cell antigen receptors is their inability to recognize soluble molecules—they can recognize peptide fragments of protein antigens on the cell surface bound to specialized peptide display molecules, called major histocompatibility complex (MHC). T-helper cells need MHC class II molecules for recognizing antigenic peptide fragments, and cytotoxic T-cells need MHC class I molecules. This feature enables T-cells to detect intracellular pathogens, which otherwise could remain undetected by the immune system, because short peptides (9-10 amino acids) from all proteins synthesized in eukaryotic cells (including peptides derived from pathogens) are exposed on the cell surface in the, peptide pockets' of MHC molecules. Adaptive immune response is initiated after pathogen capture by professional antigen presenting cells (APCs). Naive T-lymphocytes need to see antigens presented by MHC-antigens on APCs. These cells are present in all epithelia of the body, which is the interface between the body and external environment. In addition to that, APCs are present in smaller numbers in most other organs. APCs in the epithelia belong to the lineage of dendritic cells. In the skin, the epidermal dendritic cells are called Langerhans cells. Dendritic cells capture antigens of microbes that enter the epithelium, by the process of phagocytosis or pinocytosis. After antigen capture dendritic cells round up and lose their adhesiveness for the epithelium, they leave the epithelium and migrate via lymphatic vessels to the lymph node draining that epithelium. During the process of migration the dendritic cells mature into cells capable of stimulating T-cells. This maturation is reflected in increased synthesis and stable expression of MHC molecules, which display antigen to T-cells, and other molecules, co-stimulators, that are required for full T-cell responses. The result of this sequence of events is that the protein antigens of microbes are transported to the specific regions of lymph nodes where the antigens are most likely to encounter T-lymphocytes. Naive T-lymphocytes continuously recirculate through lymph nodes, and it is estimated that every naive T-cell in the body may cycle through some lymph nodes at least once a day. Thus, initial encounter of T-cells with antigens happens in lymph nodes and this is called priming. Primed CD4+ T-helper cells start secreting a variety of cytokines, which help other cells of the immune system to respond. Dendritic cells carry to the lymph nodes not only peptide fragments from pathogens, but also PRR-induced signals sent from innate immune system (as mentioned above, type I IFNs influence activation and differentiation of dendritic cells). Dendritic cells convert this information into activation of specific clones of T-cells (that recognize pathogenic peptides) and differentiation of suitable type of T-helper cells. Priming of CD8+ T-cells is also performed by dendritic cells, but further proliferation and maturation of CD8+ T-cells into fully functional killer cells depends on cytokines secreted by T-helper cells.
Taken together, between the innate and adaptive immune system there is a continuous and complicated interplay. Success in developing vaccines against “difficult” pathogens where no vaccines are currently available (HIV-1, TB and malaria) might depend on exploiting completely new methods for eliciting a protective immune response.
Antiviral Response to Positive-Strand RNA Viruses and their Replication By-Products
Positive-Strand RNA Viruses
Positive-strand RNA viruses encompass over one-third of all virus genera. Positive-strand RNA virus genomes are templates for both translation and replication, leading to interactions between host translation factors and RNA replication at multiple levels. All known positive-strand RNA viruses carry genes for an RNA-dependent RNA polymerase (RdRp) used in genome replication. However, unlike other RNA viruses, positive-strand RNA viruses do not encapsidate this polymerase. Thus, upon infection of a new cell, viral RNA replication cannot begin until the genomic RNA is translated to produce polymerase and, for most positive-strand RNA viruses, additional replication factors. All characterized positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. In and beyond the alphavirus-like superfamily the replication of viral RNA occurs in association with spherical invaginations of intracellular membranes. For example, alphaviruses use endosomal and lysosomal membranes for their replication complex assembly. The membrane provides a surface on which replication factors are localized and concentrated. This organization also helps to protect any dsRNA replication intermediates from dsRNA-induced host defence responses such as RNA interference or interferon-induced responses (Ahlquist P et al 2003).
Despite differences in genome organization, virion morphology and host range, positive-strand RNA viruses have fundamentally similar strategies for genome replication. By definition, the viral (+)RNA genome has the same polarity as cellular mRNA and the viral genomic RNA is directly translated by the cellular translation machinery. Firstly, non-structural proteins are synthesized as precursor polyproteins and cleaved into mature non-structural proteins by viral proteases. A large part of the viral genome is devoted to non-structural proteins, which are not part of the virion and carry out important functions during viral replication. Following translation and polyprotein processing, a complex is assembled that includes the RdRp, further accessory non-structural proteins, viral RNA and host cell factors. These so-called replication complexes (RCs) carry out viral-RNA synthesis. Negative-sense viral RNA is synthesized early in infection and after the formation of replication complexes this negative-strand RNA is used as a template to synthesize full-length positive-sense genomic RNA as well as the subgenomic RNA. The key enzyme responsible for these steps is the RNA-dependent RNA-polymerase, which act within replicase complex (Moradpour et al 2007, Miller and Krijnse-Locker 2008).
Viral RNA Sensing
Positive strand RNA viruses produce in the process of replication negative strand RNA, positive strand RNA, double strand RNA (dsRNA) and subgenomic mRNA, which are themselves powerful inducers of innate immune response pathways. The effect is induced through TLR3 (dsRNA), TLR7/8 (ssRNA), and some other TLRs which recognize the specific structural elements in the secondary structure of the ssRNA. For example, positive strand RNA virus, yellow fever virus live attenuated vaccine is definitely one of the most effective vaccines available that activates innate immunity via multiple Toll-like receptors which also induces differential effects on the quality of the long-lasting antigen-specific T cell response (Querec TD and Pulendran B Adv Exp Med. Biol. 2007; 590:43-53).
As stated above, cells possess receptors and signalling pathways to induce antiviral gene expression in response to cytosolic viral presence. Multiple cytokines are induced by virus infection including interleukine-6 (IL-6), IL-12 p40, and tumor necrosis factor (TNF), but the hallmark of antiviral responses is the production of type I interferons. Type I interferons include multiple subtypes encoded by separate intronless genes: one IFN-β and 13-14 IFN-α subtypes, depending on species. Type I interferons can be produced by all nucleated cells, including epithelial cells, fibroblasts at mucosal surfaces, and dendritic cells, in response to virus infection. In addition all cells can respond to type I interferons through the type I interferon receptor (IFNAR), which binds all subtypes.
Genes encoding the cytosolic PRRs and the components of the downstream signalling pathways are themselves interferon inducible, leading to a positive-feedback loop that can greatly amplify innate antiviral responses. It has been thought that this loop is set in motion by the presence of dsRNA in cells. dsRNA fulfills the criteria for being a marker of virus infection, as long dsRNA molecules are absent from uninfected cells but can be formed by the complementary annealing of two strands of RNA produced during the replication of RNA viruses. dsRNA is known to activate nuclear factor kappa B (NF-κB) and interferon regulatory factors-3 (IRF-3) and -7, that are essential in the synthesis of type I IFNs. Interferons mediate their antiviral response via specific cell surface receptors, IFNAR, that activate cytoplasmic signal transducers and activators of transcription (STATs), which translocate into the nucleus and activate numerous IFN-stimulated genes (ISGs) (Rautsi et al 2007).
Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytoplasmic IFN-inducible DExD/H box RNA-helicases that can detect intracellular viral products, such as genomic RNA, and signal for IRF3 and IRF7 activation and for the induction of IFN-α, -β, and -λ gene expression. RIG-I is a cytosolic protein containing RNA-binding helicase domain and two caspase activation and recruitment domains (CARDS). Like RIG-I, MDA5 bears a RNA-helicase domain and two CARDs. They both signal through interferon-β promoter stimulator-1 (IPS-1). Signal adaptor IPS-1 is located on mitochondria and contains an N-terminal CARD that forms homotypic interactions with CARDs of RIG-I and Mda5. This results in activation of the C-terminal catalytic domain and the initiation of a signalling cascade that culminates in the transcription of cytokine genes through activation of NF-κB and IRF3.
Although both RIG-I and Mda5 bind poly(I:C), a synthetic dsRNA, and signal via a common pathway, they selectively respond to different viruses. For example RIG-I detects influenza A virus, vesicular stomatitis virus (VSV), Japanese encephalitis virus (JEV), and Sendai virus (SeV), whereas MDA5 detects picornaviruses, such as encephalomyocarditis virus (EMCV), Theiler's encephalomyelitis virus, and mengovirus. Independently of single or double strandedness the critical element in RIG-I stimulation by RNA is the presence of 5′-triphosphates. Which also provides explanation for the virus specificity of RIG-I.
Type I interferons affect various subtypes of dendritic cells (DCs). They can act as an autocrine survival factors for certain natural interferon producing cells, promote the differentiation of peripheral blood monocytes to DCs and induce their phenotypic and functional maturation. As most cell types are capable of expressing type I interferons, maturation of DCs in non-lymphoid tissues may be triggered following infection of neighbouring cells. These DCs will acquire the ability to migrate to lymphoid organs and initiate T cell responses (LeBon and Tough 2002).
Type I interferon signalling also upregulates IFN-γ production by DCs and T cells and thereby favours the induction and maintenance of Th1 cells. Additionally, acting directly or indirectly, they can influence the expression and function of a variety of cytokines. For example enhance interleukin-6 (IL-6) signalling, and production of anti-inflammatory transforming growth factor β (TGF-β), IL-1 receptor antagonist and soluble tumor necrosis factor (TNF) receptors. Type I interferons or their inducers can also elicit high IL-15 expression by DCs, thereby causing strong and selective stimulation of memory-phenotype CD8+ T cells (Theofilopoulos et al 2005).
Specific viral pathogen infection related patterns (like accumulation of the dsRNA in cytoplasm of the virus infected cells), recognition factors responding to these patterns (e.g. Toll-like receptors), and different anti-viral defence pathways triggered by these interactions have been described above. The complex system called innate immunity is directed to lead the cascade of events from recognition of pathogen to destroying the virus infected cells and rapid clearing of the virus infection from the body. In addition, the activation of the innate immune system is an important determinant of the quantity and quality of the adaptive immune response evoked against the viral antigens (Germain RN 2004).
Immunological Adjuvants.
Immunological adjuvants were originally described by Ramon in 1924 as substances used in combination with a specific antigen that produced a more robust immune response than the antigen alone. This very broad definition includes a wide variety of materials. The immunological adjuvants available today fall broadly into two categories: delivery systems and immune potentiators (for review Fraser C. K., Diener K. R., Brown M. P. and Hayball J. D. (2007) Expert Reviews in Vaccines 6(4)559-578).
Delivery systems can change the presentation of the antigen within the vaccine thus maximizing antigen exposure to the immune system, targeting antigen in a certain form to specific physiological locations thereby assuring pick-up of the antigen by the professional Antigen Presenting Cells (APCs). Examples of immunological adjuvants presented as delivery system type adjuvants in the formulations of vaccines are alum, emulsions, saponins and cationic lipids.
Immune activators act directly on immune cells by activating the pathways significant for induction of adaptive immunity. These may be exogenous microbial or viral components, their synthetic derivatives or endogenous immunoactive compounds such as cytokines, chemokines and costimulatory molecules. This type of molecules can enhance specific immunity to the target antigen. As of today, toll-like receptor agonists, nucleotide oligomerization domain-like receptor agonists, recombinant endogeneous compounds like cytokines, chemokines or costimulatory molecules are available and may serve as immune potentiators. It is however important to emphasize that cytokines and chemokines are species-specific molecules and therefore are not readily comparable in different animals. In these cases the homologues of respective molecules need to be used, which considerably complicates the use of such adjuvants as well as the interpretation of experimental results in one species and the extrapolation thereof to another species.
DNA vaccines as several other genetic vaccines have been developed over several years and present a promising approach in the induction of specific immune responses in test animals. However, these vaccines have turned out to be ineffective in humans and larger animals. One of the reasons is probably that the reactivity and immunogenicity is lower than for traditional vaccines. A likely reason for this deficiency is the limited capacity for protein expression in vivo, which is of greater significance in outbred animals, including humans as well as the more homogeneous nature and lack of contaminating pathogen-derived ingredients in the actual vaccine preparation.
This has caused a need for the development of specific, finely tuned immunological adjuvants for the preparation of vaccines, which would be targeted for activation of the immune system without profound toxic effects. As a result of this need, efforts have been made to combine DNA vaccines with cytokines or chemokines, like hematopoietic growth factors, such as GM-CSF, or chemokines like MIP-1α, which can improve the immune responses against the antigen encoded by the DNA vaccine. However, unfortunately these effects are still quite weak. Co-delivery of the cytokines and chemokines as proteins requires enormous work before a good quality protein can be produced for actual use in animals or humans.
As for the use of nucleic acid based expression vectors for the expression of an adjuvant for use in combination with DNA vaccines, questions arise regarding the appropriate level and site of expression of a particular adjuvant molecule and the effect of this expression on the tissue to which the vaccine is administered.
The observations about the potential useful effect of adjuvants in immune stimulation were made in the early days by Gaston Ramon who found that higher antibody titers were developed in the horses which developed abscesses post-vaccination. The concept of using immunological adjuvants to improve antigen-specific immune responses has been inseparably linked from the early findings with their capacity to induce inflammatory processes due to contaminations. As a result, the use of such immunological adjuvants may cause clinically unacceptable toxicity and serious health concerns. Therefore, the only globally licensed adjuvant for human use is alum, a weak adjuvant capable only of inducing humoral immunity. All the other stronger adjuvants capable of inducing both humoral and cell-mediated immunity available today are confined to experimental use only.
It has been shown that the current repertoire of vaccine adjuvants is inadequate to generate effective vaccines against significant pathogens including HIV1, malaria and tuberculosis (Riedmann et al. 2007; Fraser et al. 20007). Combination of known adjuvants may overcome some of the problems associated with the vaccines that are available, however, a reliable, safe and advanced new generation of immune modulators in the form of adjuvants is certainly needed.
In view of the problems still present in the prior art explained in the above, the aim of the present invention was thus to find a more efficient adjuvant to accompany and improve the responses to vaccines available today. The adjuvant according to the present invention, is a modulator of the immune system, meaning that it will improve and strengthen the immune response in a subject to whom the vaccine is administered.